Endo-beta-manna hydrolase Man01929 and method for mutating endo-beta-manna hydrolase Man01929 into glycosyl transferase and application of endo-beta-manna hydrolase Man01929

A mannanase and mutant enzyme technology, applied in the directions of transferase, hydrolase, glycosylase, etc., can solve the problem of little research on the mechanism, and achieve the effect of stable physical and chemical properties and wide pH tolerance

Active Publication Date: 2020-07-28
JINAN ACTIVE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, β-mannanase itself has a flexible structure and can recognize and bind a variety of substrates, but there are few mechanistic studies on which glycosyl binding sites are specifically involved in the selective recognition and action process of substrates
[0008] It has been reported in the literature above that this ability to transglycosylate is related to tryptophan (Trp) at the +2 subsite of β-mannanase, but whether amino acid residues at other subsites are involved in this regulatory mechanism, and Whether other types of amino acid residues are involved has not been reported

Method used

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  • Endo-beta-manna hydrolase Man01929 and method for mutating endo-beta-manna hydrolase Man01929 into glycosyl transferase and application of endo-beta-manna hydrolase Man01929
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  • Endo-beta-manna hydrolase Man01929 and method for mutating endo-beta-manna hydrolase Man01929 into glycosyl transferase and application of endo-beta-manna hydrolase Man01929

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Extraction of Genomic DNA from Pyrobacterium pyrobacter MY04 Strain

[0088] Pyrochrome bacterium MY04 was inoculated into liquid medium YT04, and cultured with shaking at 28°C and 200 rpm until its absorbance at 600 nm (OD 600 ) is 1.2; take 10mL of the cultured bacteria solution, and centrifuge at 4°C for 15min under the condition of 12,000×g (g, the gravitational constant of the earth) to collect the bacterial precipitate; Suspend the bacteria, centrifuge at 12,000x g, 4°C for 15 minutes, and collect the bacteria pellet.

[0089] The above-mentioned liquid medium YT04 has the following components per liter:

[0090] Tryptone 10g, yeast extract 5.0g, sodium chloride 30g, dissolved in water and adjusted to 1L, pH 7.2.

[0091] Add 6.0mL of lysozyme buffer solution to each tube to obtain about 7.0mL of bacterial solution, and add 280μL of lysozyme solution with a concentration of 20mg / mL to make the final concentration of lysozyme 800μg / mL; Place in an ice-water bath...

Embodiment 2

[0093] Genome Scanning and Sequence Analysis of Pyrobacterium pyrobacter MY04 Strain

[0094] Genomic DNA prepared in Example 1 was scanned and sequenced by Shanghai Meiji Biotechnology Co., Ltd. using pyrosequencing technology. The DNA sequencing results were analyzed with the online software of NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ) website. The analysis software used on the NCBI website is Open Reading Frame Finder (ORF Finder, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0095] The results analyzed by the above-mentioned biological software show that the genomic DNA of the Pyrobacterium pyrobacterium MY04 strain carries a coding gene man01929 of β-mannanase, the coding region of the gene man01929 is 2799bp long, and the nucleotide sequence is shown in SEQ ID NO.1 . The β-mannanase Man01929 encoded by the gene man01929 contains a total of ...

Embodiment 3

[0098] Recombinant expression of gene man01929 in Escherichia coli BL21 (DE3) bacterial strain:

[0099] Using the genomic DNA prepared in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:

[0100] forward primer for man01929 amplification

[0101] Man01929-F: 5'-gcg CATATG GCACTTTTTGCTCATGC-3';

[0102] reverse primer for man01929 amplification

[0103] Man01929-F: 5'-gcg CTCGAG TTGCTTGTAGATTCTCCTAAC-3';

[0104] The underlined mark of the forward primer is the specificity site of the restriction endonuclease Nde I, and the underlined mark of the reverse primer is the specificity site of the restriction endonuclease Xho I.

[0105] The high-fidelity DNA polymerase Prime STAR HS DNA Polymerase used was purchased from China Dalian Bao Biological Company, and the PCR reaction reagents used were operated according to the product instructions provided by the company.

[0106] PCR reaction system:

[0107] 2×Primer star GC buff...

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Abstract

The invention relates to an endo-beta-manna hydrolase Man01929 and a method for mutating the endo-beta-manna hydrolase Man01929 into glycosyl transferase and application of the endo-beta-manna hydrolase Man01929; an amino acid sequence of the endo-beta-manna hydrolase Man01929 is shown by SEQ ID NO.2; and a nucleotide sequence encoding the Man01929 is shown by SEQ ID NO.1. Site-directed mutation is performed on the hydrolase, and a result shows that 118, 119, 124 and 323 sites of the amino acid sequence play an important role in the substrate selective action process of recognition, binding, degradation and the like of the Man01929 on galactomannan, and the shown result has reference value for promoting research of the substrate selectivity mechanism of GH5 family beta-mannase. In addition, the invention further obtains a mutant D124Y with transglycosylation ability, and thus, an important reference is provided for revealing the transformation mechanism of the catalysismechanism of transforming the hydrolase into the glycosyl transferase.

Description

technical field [0001] The invention relates to an endo-type β-mannan hydrolase Man01929 and a method for mutating it into a glycosyltransferase and its application, which belongs to the field of biotechnology and relates to protein improvement technology. Background technique [0002] Mannan is a kind of polysaccharide with complex structure, which can be divided into four types according to the structure of sugar units: pure mannan, glucomannan, galactomannan ) and galactoglucomannan (galactoglucomannan). Due to the complex structure of mannan, the synergy of various enzymes is required for complete hydrolysis, such as β-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), β-glucosidase (EC 3.2.1.21), acetylmannan esterase (EC 3.1.1.6), alpha-galactosidase (EC 3.2.1.22). β-mannanase is a kind of glycosyl hydrolase (Glycoside Hydrolase, GH), which plays a key role in the hydrolysis process. It can hydrolyze the β-1,4 glycosidic bonds inside the main chain of mannan to pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/24C12N9/10C12P19/14C12P19/00
CPCC12N9/1048C12N9/2494C12P19/00C12P19/14C12Y302/01078
Inventor 韩文君程媛媛宓延红古静燕李新卫洁
Owner JINAN ACTIVE BIOTECHNOLOGY CO LTD
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